Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity

Abstract

Background: Dengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking.

Methodology/principal findings: In this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the "early" group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0-1 and declined on day 3-4; the second "late" group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5-6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0-3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1.

Conclusions/significance: Our study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1-3 may have applications in developing early molecular diagnostics for DHF.

Figure 1. Daily gene expression patterns define 2 subtle phases during the disease process.

a) Illness days with corresponding stages (G) and phases for samples on the HG-focus platform. The number of febrile and defervescent samples in each G was shown. b) A global overview of the gene expression pattern in G0 to G7. Using convalescent samples (G7) as a baseline, significant genes in each stage (from G0 to G6) were detected using multi-way ANOVA. PCA was performed using all of the significant genes found in G0 to G7 on the HG-focus platform. All of the samples (represented by dots) were included in the PCA. c) PCA was performed on the defervescent samples only. d) PCA was performed on the febrile samples only and are indicated according to the number of fever days. e) Samples were reassigned to the early acute, late acute and convalescent phases. Differentially expressed genes in the early acute vs. convalescent or late acute vs. convalescent phase were detected using multi-way ANOVA. Shared genes between the early and late acute phases were represented by a Venn diagram. f) PCA was performed using 313 phase-specific genes.

Figure 2. 2-level and 1-level cross-validation identified a top classification model and 65 gene signatures for the prediction of 3 phases.

a) Information of the model selection showing 65 variables and a top classification model with 2-level and 1-level normalized correction rates. b) Heatmap of the 65 signatures.

Figure 3. Two waves of gene expression over the disease stages.

Median marker expression intensity (log 2 value) of the genes detected in the early and late acute phases from G0 to G7 was shown.

Figure 4. Classification of DF and DHF.

a) The expression pattern of the top 7 genes (selected from 140 genes) is indicated using PCA. b) The dynamic expression (shown by median Log2 gene expression intensity) of 7 DF-DHF signatures from G1 to G7.